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Blood, Vol. 114, Issue 1, 105-108, July 2, 2009
Early defects in human T-cell development severely affect distribution and maturation of thymic stromal cells: possible implications for the pathophysiology of Omenn syndrome
Blood Poliani et al.
114: 105
Supplemental materials for: Poliani et al
Single-staining immunohistochemical procedure
Three µm sections were taken from formalin-fixed, paraffin-embedded blocks and subjected to routine hematoxylin and eosin (H&E) staining and immunohistochemical analysis. Briefly, sections were de-waxed, re-hydratated and endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol. Epitope retrieval was performed for all primary antibodies used (with the exception for the anti-human S100) by either microwave treatment in 1.0 mM EDTA buffer pH 8.0 or by incubating in a thermostatic bath for 40′ at 98°C. Sections were then cooled down, washed in TRIS-base buffer at pH 7.4, pre-incubated in blocking buffer containing 5% normal goat serum in Tris-HCl, and then incubated for 1 hours with primary antibody in Tris/1% Bovine Serum Albumin (BSA). Sections were then washed in Tris-HCl buffer prior to incubation for 30 minutes with the secondary antibody (ChemMATE Envision Rabbit/Mouse, DAKO Cytomation, Glostrup Denmark) or with super-sensitive immunohistochemistry detection system (Biogenex, San Ramon, CA, USA). Signal was revealed by either diaminobenzydine (DAB) (brown colour) or Fast Red (DAKO) (red colour), and slides were counterstained with Hematoxylin. Maturation of medullary thymic epithelial cells (mTECs) was assessed by staining thymic sections with rabbit polyclonal anti-human claudin-4 (1:100; Zymed Laboratories, San Francisco, CA, USA), monoclonal mouse anti-human Aire (1:5000; kindly provided by Prof. P. Peterson, University of Tartu, Estonia) and biotin-conjugated Ulex Europaeus Agglutinin-1 (UEA-1) (1:600; Vector Laboratories, Burlingame, CA, USA). The presence of dendritic cells was assessed by monoclonal mouse–anti-human CD208 (DC-LAMP) (1:100; clone 104.G4, Immunotech, Marseille, France), monoclonal mouse anti-human CD11c (1:30; Novocastra Laboratories LTD, United Kingdom), monoclonal mouse anti-CD303 (BDCA2) (1:50; Dendritics, Lyon, France) and polyclonal rabbit anti-human S100 (1:5000; Dako Cytomation, Glostrup, Denmark) Finally, development of natural regulatory T cells (nTreg) was studied by staining sections with monoclonal rat anti-human Foxp3 antibody (1:200; eBioscience, San Diego, CA, USA). Double-staining immunofluorescence procedure
Differential expression of cytokeratin 5 (CK5) and CK8 was assessed by double-staining immunofluorescence procedure using rabbit polyclonal anti-CK5 (1:50; Covance, Berkeley, CA, USA) and anti-CK8 rat monoclonal antibody (1:200; clone TROMA-1, kindly provided by Prof. P. Brulet, Institut Pasteur, Paris Cedex, France). Briefly, sections were de-waxed, re-hydratated, and epitope retrieval was performed for both primary antibodies by means of microwave treatment in 1.0 mM EDTA buffer pH 8.0. Sections were then cooled down, washed in a TRIS-base buffer at pH 7.4, pre-incubated in blocking buffer containing 5% normal serum (specific for the secondary antibody) in Tris-HCl for 5 minute, and incubated for 1 hours with CK5 and CK8 primary antibodies in Tris/1% Bovine Serum Albumin (BSA). Signal was revealed by using secondary swine anti-rabbit FITC-conjugated antibody (1:30; Dako Cytomation, Glostrup, Denmark) for CK5 and rabbit anti-rat biotinylated antibody (1:200; Vector Laboratories, Burlingham, CA, USA) followed by Streptavidin-Texas Red (1:100; Southern Biotechnology Associates, Birmingham, AL, USA) for CK8. Sections were then counter-stained with DAPI. Bright field double-staining immunohistochemical procedure
The same procedures described in the single staining immunohistochemical procedure paragraph have been applied prior to the incubation with the specific first primary antibody for 1 hour. Sections were then washed in Tris-HCl buffer prior to incubation for 30 minutes with the first secondary antibody HRP conjugated (ChemMATE Envision Rabbit/Mouse, DAKO Cytomation, Glostrup Denmark) and signal has been revealed with diaminobenzydine (DAB). Sections were then washed in Tris-HCl buffer prior to incubation with the second primary antibody for 1 hour, wash again in the same buffer and incubated for 30 minutes with the specific secondary biotinylated secondary antibody followed by incubation for 20 minutes in Alkaline Phosphatase-conjugated Strepavidin. Signal has been revealed by Ferangi Blue Chromogen Kit (Biocare Medical, Concord, CA, USA) and nuclei counterstained with Methyl Green. The following antibodies have been used: monoclonal mouse anti-human CD163 (1:50, clone 10D6, Thermoscientific, Fremont, CA, USA), monoclonal mouse anti-human CD11c (1:30; Novocastra Laboratories LTD, United Kingdom), monoclonal mouse–anti-human CD208 (DC-LAMP) (1:100; clone 104.G4, Immunotech, Marseille, France) and monoclonal rat anti-human Foxp3 antibody (1:200; eBioscience, San Diego, CA, USA).
Files in this Data Supplement:
- Figure S1. Early and severe defects that severely compromize T-cell development result in profound abnormalities of thymic compartmentalization, with impaired maturation of mTEC, dendritic cells and natural regulatory T cells (JPG, 0.97 MB)
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The thymic biopsy from a control subject (Panel A) shows normal thymic architecture with CMD (H&E staining), normal distribution of CK8+CK5− cTECs (CK8, red) and CK8−CK5+ mTECs (CK5, green) and expression of Cld4 and Aire (brown staining, upper and lower images, respectively). Thymic biopsies from patients with genetic defects that are non permissive (ADA deficiency and CD3δ deficiency, Panels B and C) or that are partially permissive for T-cell development (OS due to RAG2 mutations, Panel D), show profound abnormalities of the thymic architecture, with severe atrophy and lobular pattern (H&E staining), loss of CMD, presence of a diffuse epithelial network of CK5+CK8+ immature TECs (yellow) and no expression of Cld4 and Aire. The control thymus shows normal distribution of both S-100+ and CD11c+ dendritic cells (Panel A, CD11c+ upper image, red staining; S-100+ lower image, brown staining) along with the presence of Foxp3+ nTreg interacting with mature activated CD208+ (DC-LAMP) DCs (Panel F). In contrast, severe depletion of thymic Foxp3+ nTregs and dendritic cells (as shown by staining for DC-LAMP and S-100) is present in thymic biopsies from patients with SCID or with OS (Panels B–D). In these biopsies, double staining for CD11c and CD163 shows that most CD11c+ cells co-express CD163, suggesting their macrophage nature. H&E staining, 10× original magnification; Immunofluorescence stainings, 20× original magnification: CK5 (green), CK8 (red), nuclei (blue), merge (yellow). Single immunohistochemical stainings, 40× original magnification: Cld4, Aire, S-100 (brown) and CD11c (red); Double immunohistochemical stainings, 40× original magnification: CD11c (blue) and CD163 (brown); Foxp3 (blue) and DC-LAMP (CD208) (brown).
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